BRAFV600E-Mutant Metastatic Colorectal Cancer: Current Evidence, Future Directions, and Research Priorities.

BRAFV600E-mutant metastatic colorectal cancer represents a distinct molecular phenotype known for its aggressive biological behavior, resistance to standard therapies, and poor survival rates. Improved understanding of the biology of the BRAF oncogene has led to the development of targeted therapies that have paved the way for a paradigm shift in managing this disease. However, despite significant recent advancements, responses to targeted therapies are short-lived, and several challenges remain. In this review, we discuss how progress in treating BRAFV600E-mutant metastatic colorectal cancer has been made through a better understanding of its unique biological and clinical features. We provide an overview of the evidence to support current treatment approaches and discuss critical areas of need and future research strategies that hold the potential to refine clinical practice further. We also discuss some challenging aspects of managing this disease, particularly the complexity of acquired resistance mechanisms that develop under the selective pressure of targeted therapies and rational strategies being investigated to overcome them.

BCL-XL inhibitors enhance the apoptotic efficacy of BRAF inhibitors in BRAFV600E colorectal cancer.

Metastatic BRAFV600E colorectal cancer (CRC) carries an extremely poor prognosis and is in urgent need of effective new treatments. While the BRAFV600E inhibitor encorafenib in combination with the EGFR inhibitor cetuximab (Enc+Cet) was recently approved for this indication, overall survival is only increased by 3.6 months and objective responses are observed in only 20% of patients. We have found that a limitation of Enc+Cet treatment is the failure to efficiently induce apoptosis in BRAFV600E CRCs, despite inducing expression of the pro-apoptotic protein BIM and repressing expression of the pro-survival protein MCL-1. Here, we show that BRAFV600E CRCs express high basal levels of the pro-survival proteins MCL-1 and BCL-XL, and that combining encorafenib with a BCL-XL inhibitor significantly enhances apoptosis in BRAFV600E CRC cell lines. This effect was partially dependent on the induction of BIM, as BIM deletion markedly attenuated BRAF plus BCL-XL inhibitor-induced apoptosis. As thrombocytopenia is an established on-target toxicity of BCL-XL inhibition, we also examined the effect of combining encorafenib with the BCL-XL -targeting PROTAC DT2216, and the novel BCL-2/BCL-XL inhibitor dendrimer conjugate AZD0466. Combining encorafenib with DT2216 significantly increased apoptosis induction in vitro, while combining encorafenib with AZD0466 was well tolerated in mice and further reduced growth of BRAFV600E CRC xenografts compared to either agent alone. Collectively, these findings demonstrate that combined BRAF and BCL-XL inhibition significantly enhances apoptosis in pre-clinical models of BRAFV600E CRC and is a combination regimen worthy of clinical investigation to improve outcomes for these patients.

Type of anesthesia for cancer resection surgery: No differential impact on cancer recurrence in mouse models of breast cancer.

BACKGROUND: Surgery is essential for curative treatment of solid tumors. Evidence from recent retrospective clinical analyses suggests that use of propofol-based total intravenous anesthesia during cancer resection surgery is associated with improved overall survival compared to inhaled volatile anesthesia. Evaluating these findings in prospective clinical studies is required to inform definitive clinical guidelines but will take many years and requires biomarkers to monitor treatment effect. Therefore, we examined the effect of different anesthetic agents on cancer recurrence in mouse models of breast cancer with the overarching goal of evaluating plausible mechanisms that could be used as biomarkers of treatment response. METHODS: To test the hypothesis that volatile anesthesia accelerates breast cancer recurrence after surgical resection of the primary tumor, we used three mouse models of breast cancer. We compared volatile sevoflurane anesthesia with intravenous propofol anesthesia and used serial non-invasive bioluminescent imaging to track primary tumor recurrence and metastatic recurrence. To determine short-term perioperative effects, we evaluated the effect of anesthesia on vascular integrity and immune cell changes after surgery in animal models. RESULTS: Survival analyses found that the kinetics of cancer recurrence and impact on survival were similar regardless of the anesthetic agent used during cancer surgery. Vascular permeability, immune cell infiltration and cytokine profiles showed no statistical difference after resection with inhaled sevoflurane or intravenous propofol anesthesia. CONCLUSIONS: These preclinical studies found no evidence that choice of anesthetic agent used during cancer resection surgery affected either short-term perioperative events or long-term cancer outcomes in mouse models of breast cancer. These findings raise the possibility that mouse models do not recapitulate perioperative events in cancer patients. Nonetheless, the findings suggest that future evaluation of effects of anesthesia on cancer outcomes should focus on cancer types other than breast cancer.

Notch receptor/ligand diversity: contribution to colorectal cancer stem cell heterogeneity.

Cancer cell heterogeneity is a key contributor to therapeutic failure and post-treatment recurrence. Targeting cell subpopulations responsible for chemoresistance and recurrence seems to be an attractive approach to improve treatment outcome in cancer patients. However, this remains challenging due to the complexity and incomplete characterization of tumor cell subpopulations. The heterogeneity of cells exhibiting stemness-related features, such as self-renewal and chemoresistance, fuels this complexity. Notch signaling is a known regulator of cancer stem cell (CSC) features in colorectal cancer (CRC), though the effects of its heterogenous signaling on CRC cell stemness are only just emerging. In this review, we discuss how Notch ligand-receptor specificity contributes to regulating stemness, self-renewal, chemoresistance and cancer stem cells heterogeneity in CRC.

Use of patient-derived explants as a preclinical model for precision medicine in colorectal cancer: A scoping review.

PURPOSE: Whilst the treatment paradigm for colorectal cancer has evolved significantly over time, there is still a lack of reliable biomarkers of treatment response. Treatment decisions are based on high-risk features such as advanced TNM stage and histology. The role of the tumour microenvironment, which can influence tumour progression and treatment response, has generated considerable interest. Patient-derived explant cultures allow preservation of native tissue architecture and tumour microenvironment. The aim of the scoping review is to evaluate the utility of patient-derived explant cultures as a preclinical model in colorectal cancer. METHODS: A search was conducted using Ovid MEDLINE, EMBASE, Web of Science, and Cochrane databases from start of database records to September 1, 2022. We included all peer-reviewed human studies in English language which used patient-derived explants as a preclinical model in primary colorectal cancer. Eligible studies were grouped into the following categories: assessing model feasibility; exploring tumour microenvironment; assessing ex vivo drug responses; discovering and validating biomarkers. RESULTS: A total of 60 studies were eligible. Fourteen studies demonstrated feasibility of using patient-derived explants as a preclinical model. Ten studies explored the tumour microenvironment. Thirty-eight studies assessed ex vivo drug responses of chemotherapy agents and targeted therapies. Twenty-four studies identified potential biomarkers of treatment response. CONCLUSIONS: Given the preservation of tumour microenvironment and tumour heterogeneity, patient-derived explants has the potential to identify reliable biomarkers, treatment resistance mechanisms, and novel therapeutic agents. Further validation studies are required to characterise, refine and standardise this preclinical model before it can become a part of precision medicine in colorectal cancer.

S100 family proteins are linked to organoid morphology and EMT in pancreatic cancer.

Epithelial-mesenchymal transition (EMT) is a continuum that includes epithelial, partial EMT, and mesenchymal states, each of which is associated with cancer progression, invasive capabilities, and ultimately, metastasis. We used a lineage-traced sporadic model of pancreatic cancer to generate a murine organoid biobank from primary and secondary tumors, including sublines that underwent partial EMT and complete EMT. Using an unbiased proteomics approach, we found that organoid morphology predicts the EMT state, and the solid organoids are associated with a partial EMT signature. We also observed that exogenous TGFß1 induces solid organoid morphology that is associated with changes in the S100 family, complete EMT, and the formation of high-grade tumors. S100A4 may be a useful biomarker for predicting EMT state, disease progression, and outcome in patients with pancreatic cancer.

Computational Screening of Anti-Cancer Drugs Identifies a New BRCA Independent Gene Expression Signature to Predict Breast Cancer Sensitivity to Cisplatin.

The development of therapies that target specific disease subtypes has dramatically improved outcomes for patients with breast cancer. However, survival gains have not been uniform across patients, even within a given molecular subtype. Large collections of publicly available drug screening data matched with transcriptomic measurements have facilitated the development of computational models that predict response to therapy. Here, we generated a series of predictive gene signatures to estimate the sensitivity of breast cancer samples to 90 drugs, comprising FDA-approved drugs or compounds in early development. To achieve this, we used a cell line-based drug screen with matched transcriptomic data to derive in silico models that we validated in large independent datasets obtained from cell lines and patient-derived xenograft (PDX) models. Robust computational signatures were obtained for 28 drugs and used to predict drug efficacy in a set of PDX models. We found that our signature for cisplatin can be used to identify tumors that are likely to respond to this drug, even in absence of the BRCA-1 mutation routinely used to select patients for platinum-based therapies. This clinically relevant observation was confirmed in multiple PDXs. Our study foreshadows an effective delivery approach for precision medicine.

Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy.

Tumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

Longitudinal Monitoring of Intra-Tumoural Heterogeneity Using Optical Barcoding of Patient-Derived Colorectal Tumour Models.

Geno- and phenotypic heterogeneity amongst cancer cell subpopulations are established drivers of treatment resistance and tumour recurrence. However, due to the technical difficulty associated with studying such intra-tumoural heterogeneity, this phenomenon is seldom interrogated in conventional cell culture models. Here, we employ a fluorescent lineage technique termed "optical barcoding" (OBC) to perform simultaneous longitudinal tracking of spatio-temporal fate in 64 patient-derived colorectal cancer subclones. To do so, patient-derived cancer cell lines and organoids were labelled with discrete combinations of reporter constructs, stably integrated into the genome and thus passed on from the founder cell to all its clonal descendants. This strategy enables the longitudinal monitoring of individual cell lineages based upon their unique optical barcodes. By designing a novel panel of six fluorescent proteins, the maximum theoretical subpopulation resolution of 64 discriminable subpopulations was achieved, greatly improving throughput compared with previous studies. We demonstrate that all subpopulations can be purified from complex clonal mixtures via flow cytometry, permitting the downstream isolation and analysis of any lineages of interest. Moreover, we outline an optimized imaging protocol that can be used to image optical barcodes in real-time, allowing for clonal dynamics to be resolved in live cells. In contrast with the limited intra-tumour heterogeneity observed in conventional 2D cell lines, the OBC technique was successfully used to quantify dynamic clonal expansions and contractions in 3D patient-derived organoids, which were previously demonstrated to better recapitulate the heterogeneity of their parental tumour material. In summary, we present OBC as a user-friendly, inexpensive, and high-throughput technique for monitoring intra-tumoural heterogeneity in in vitro cell culture models.

A new natural killer cell-specific gene signature predicting recurrence in colorectal cancer patients.

The protective role of Natural Killer (NK) cell tumour immunosurveillance has long been recognised in colorectal cancer (CRC). However, as most patients show limited intra-tumoral NK cell infiltration, improving our ability to identify those with high NK cell activity might aid in dissecting the molecular features which underlie NK cell sensitivity. Here, a novel CRC-specific NK cell gene signature that infers NK cell load in primary tissue samples was derived and validated in multiple patient CRC cohorts. In contrast with other NK cell gene signatures that have several overlapping genes across different immune cell types, our NK cell signature has been extensively refined to be specific for CRC-infiltrating NK cells. The specificity of the signature is substantiated in tumour-infiltrating NK cells from primary CRC tumours at the single cell level, and the signature includes genes representative of NK cells of different maturation states, activation status and anatomical origin. Our signature also accurately discriminates murine NK cells, demonstrating the applicability of this geneset when mining datasets generated from preclinical studies. Differential gene expression analysis revealed tumour-intrinsic features associated with NK cell inclusion versus exclusion in CRC patients, with those tumours with predicted high NK activity showing strong evidence of enhanced chemotactic and cytotoxic transcriptional programs. Furthermore, survival modelling indicated that NK signature expression is associated with improved survival outcomes in CRC patients. Thus, scoring CRC samples with this refined NK cell signature might aid in identifying patients with high NK cell activity who could be prime candidates for NK cell directed immunotherapies.

CD44v6 Defines a New Population of Circulating Tumor Cells Not Expressing EpCAM.

Circulating tumor cells (CTCs) are promising diagnostic and prognostic tools for clinical use. In several cancers, including colorectal and breast, the CTC load has been associated with a therapeutic response as well as progression-free and overall survival. However, counting and isolating CTCs remains sub-optimal because they are currently largely identified by epithelial markers such as EpCAM. New, complementary CTC surface markers are therefore urgently needed. We previously demonstrated that a splice variant of CD44, CD44 variable alternative exon 6 (CD44v6), is highly and specifically expressed by CTC cell lines derived from blood samples in colorectal cancer (CRC) patients. Two different approaches-immune detection coupled with magnetic beads and fluorescence-activated cell sorting-were optimized to purify CTCs from patient blood samples based on high expressions of CD44v6. We revealed the potential of the CD44v6 as a complementary marker to EpCAM to detect and purify CTCs in colorectal cancer blood samples. Furthermore, this marker is not restricted to colorectal cancer since CD44v6 is also expressed on CTCs from breast cancer patients. Overall, these results strongly suggest that CD44v6 could be useful to enumerate and purify CTCs from cancers of different origins, paving the way to more efficacious combined markers that encompass CTC heterogeneity.

The Diverse Applications of Pancreatic Ductal Adenocarcinoma Organoids.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid malignancies. While immortalized cancer cell lines and genetically engineered murine models have increased our understanding of PDAC tumorigenesis, they do not recapitulate inter- and intra-patient heterogeneity. PDAC patient derived organoid (PDO) biobanks have overcome this hurdle, and provide an opportunity for the high throughput screening of potential new therapies. This review provides a summary of the PDAC PDO biobanks established to date, and discusses how they have advanced our understanding of PDAC biology. Looking forward, the development of coculturing techniques for specific immune or stromal cell populations will enable a better understanding of the crosstalk that occurs within the tumor microenvironment, and the impact of this crosstalk on treatment response.

Dependence receptors: new targets for cancer therapy.

Dependence receptors are known to promote survival and positive signaling such as proliferation, migration, and differentiation when activated, but to actively trigger apoptosis when unbound to their ligand. Their abnormal regulation was shown to be an important feature of tumorigenesis, allowing cancer cells to escape apoptosis triggered by these receptors while promoting in parallel major aspects of tumorigenesis such as proliferation, angiogenesis, invasiveness, and chemoresistance. This involvement in multiple cancer hallmarks has raised interest in dependence receptors as targets for cancer therapy. Although additional studies remain necessary to fully understand the complexity of signaling pathways activated by these receptors and to target them efficiently, it is now clear that dependence receptors represent very exciting targets for future cancer treatment. This manuscript reviews current knowledge on the contribution of dependence receptors to cancer and highlights the potential for therapies that activate pro-apoptotic functions of these proteins.

The site of breast cancer metastases dictates their clonal composition and reversible transcriptomic profile.

Intratumoral heterogeneity is a driver of breast cancer progression, but the nature of the clonal interactive network involved in this process remains unclear. Here, we optimized the use of optical barcoding to visualize and characterize 31 cancer subclones in vivo. By mapping the clonal composition of thousands of metastases in two clinically relevant sites, the lungs and liver, we found that metastases were highly polyclonal in lungs but not in the liver. Furthermore, the transcriptome of the subclones varied according to their metastatic niche. We also identified a reversible niche-driven signature that was conserved in lung and liver metastases collected during patient autopsies. Among this signature, we found that the tumor necrosis factor-α pathway was up-regulated in lung compared to liver metastases, and inhibition of this pathway affected metastasis diversity. These results highlight that the cellular and molecular heterogeneity observed in metastases is largely dictated by the tumor microenvironment.

CSK-homologous kinase (CHK/MATK) is a potential colorectal cancer tumour suppressor gene epigenetically silenced by promoter methylation.

Hyperactivation of SRC-family protein kinases (SFKs) contributes to the initiation and progression of human colorectal cancer (CRC). Since oncogenic mutations of SFK genes are rare in human CRC, we investigated if SFK hyperactivation is linked to dysregulation of their upstream inhibitors, C-terminal SRC kinase (CSK) and its homolog CSK-homologous kinase (CHK/MATK). We demonstrate that expression of CHK/MATK but not CSK was significantly downregulated in CRC cell lines and primary tumours compared to normal colonic tissue. Investigation of the mechanism by which CHK/MATK expression is down-regulated in CRC cells uncovered hypermethylation of the CHK/MATK promoter in CRC cell lines and primary tumours. Promoter methylation of CHK/MATK was also observed in several other tumour types. Consistent with epigenetic silencing of CHK/MATK, genetic deletion or pharmacological inhibition of DNA methyltransferases increased CHK/MATK mRNA expression in CHK/MATK-methylated colon cancer cell lines. SFKs were hyperactivated in CHK/MATK-methylated CRC cells despite expressing enzymatically active CSK, suggesting loss of CHK/MATK contributes to SFK hyperactivation. Re-expression of CHK/MATK in CRC cell lines led to reduction in SFK activity via a non-catalytic mechanism, a reduction in anchorage-independent growth, cell proliferation and migration in vitro, and a reduction in tumour growth and metastasis in a zebrafish embryo xenotransplantation model in vivo, collectively identifying CHK/MATK as a novel putative tumour suppressor gene in CRC. Furthermore, our discovery that CHK/MATK hypermethylation occurs in the majority of tumours warrants its further investigation as a diagnostic marker of CRC.

Survival benefit of neoadjuvant chemotherapy and surgery versus surgery first for resectable colorectal liver metastases: a cohort study.

BACKGROUND: There is continued debate about the survival benefit of neoadjuvant chemotherapy (neoCT) in patients with resectable colorectal liver metastases (CRLM). METHODS: In this retrospective cohort study, we included 201 patients with metastatic colorectal cancer who underwent their first CRLM resection and achieved resection of all sites of disease. We compared the overall survival (OS) and progression-free survival (PFS) between patients who received neoCT prior to CRLM resection with those who underwent CRLM upfront. A multivariable Cox proportional hazard regression analysis was performed to adjust for potential confounders. RESULTS: A total of 101 of 201 (51.2%) patients received chemotherapy prior to CRLM resection and 100 of 201 had surgery upfront. Multivariable Cox proportional hazard regression showed no statistically significant difference in the hazard of death for those given neoCT prior to resection of CRLM compared with surgery first for both OS and PFS (OS: hazard ratio 1.74, 95% confidence interval 0.85-3.55, P = 0.127, PFS: hazard ratio 1.42, 95% confidence interval 0.93-2.19, P = 0.107). CONCLUSION: In our series of patients with metastatic colorectal cancer who achieved surgical resection of all sites of disease, neoCT prior to CRLM resection was not associated with any survival benefit.

Towards Routine Implementation of Liquid Biopsies in Cancer Management: It Is Always Too Early, until Suddenly It Is Too Late.

Blood-based liquid biopsies are considered a new and promising diagnostic and monitoring tool for cancer. As liquid biopsies only require a blood draw, they are non-invasive, potentially more rapid and assumed to be a less costly alternative to genomic analysis of tissue biopsies. A multi-disciplinary workshop (n = 98 registrations) was organized to discuss routine implementation of liquid biopsies in cancer management. Real-time polls were used to engage with experts' about the current evidence of clinical utility and the barriers to implementation of liquid biopsies. Clinical, laboratory and health economics presentations were given to illustrate the opportunities and current levels of evidence, followed by three moderated break-out sessions to discuss applications. The workshop concluded that tumor-informed assays using next-generation sequencing (NGS) or PCR-based genotyping assays will most likely provide better clinical utility than tumor-agnostic assays, yet at a higher cost. For routine application, it will be essential to determine clinical utility, to define the minimum quality standards and performance of testing platforms and to ensure their use is integrated into current clinical workflows including how they complement tissue biopsies and imaging. Early health economic models may help identifying the most viable application of liquid biopsies. Alternative funding models for the translation of complex molecular diagnostics, such as liquid biopsies, may also be explored if clinical utility has been demonstrated and when their use is recommended in multi-disciplinary consensus guidelines.

A thiol-bound drug reservoir enhances APR-246-induced mutant p53 tumor cell death.

The tumor suppressor gene TP53 is the most frequently mutated gene in cancer. The compound APR-246 (PRIMA-1Met/Eprenetapopt) is converted to methylene quinuclidinone (MQ) that targets mutant p53 protein and perturbs cellular antioxidant balance. APR-246 is currently tested in a phase III clinical trial in myelodysplastic syndrome (MDS). By in vitro, ex vivo, and in vivo models, we show that combined treatment with APR-246 and inhibitors of efflux pump MRP1/ABCC1 results in synergistic tumor cell death, which is more pronounced in TP53 mutant cells. This is associated with altered cellular thiol status and increased intracellular glutathione-conjugated MQ (GS-MQ). Due to the reversibility of MQ conjugation, GS-MQ forms an intracellular drug reservoir that increases availability of MQ for targeting mutant p53. Our study shows that redox homeostasis is a critical determinant of the response to mutant p53-targeted cancer therapy.